Method of treating leukemia

ABSTRACT

The present invention relates to methods of treating disorders in which DOT1L-mediated protein methylation plays a part, such as cancer, by administering DOT1L inhibitor compounds and pharmaceutical compositions to subjects in need thereof.

RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S.Provisional Application No. 61/697,721, filed Sep. 6, 2012, the entirecontent of which is incorporated herein in its entirety.

BACKGROUND OF THE INVENTION

Disease-associated chromatin-modifying enzymes (e.g., DOT1L) play a rolein diseases such as proliferative disorders, metabolic disorders, andblood disorders. Thus, there is a need for the development of smallmolecules that are capable of modulating the activity of DOT1L.

SUMMARY OF THE INVENTION

The present invention provides a method for treating cancer byadministering to a subject in need thereof a therapeutically effectiveamount of a compound of Formula (I) (i.e., Compound 2) or its N-oxide ora pharmaceutically acceptable salt thereof:

where the subject had at least one prior therapy to treat ahematological cancer.

For example, the hematological cancer is refractory to the priortherapy. For example, the hematological cancer shows recurrencefollowing remission. In some embodiments, the subject received andfailed all known effective therapies for the hematological cancer.

In some embodiments, the subject has a hematological cancer selectedfrom the group consisting of acute myeloid leukemia, acute lymphoblasticleukemia, myelodysplastic syndrome, a myeloproliferative disorder, andchronic myelogenous leukemia.

In some embodiments, the subject treated by the methods of the inventionhas leukemia. For example, the leukemia is characterized by MLL generearrangement. In some embodiments, the MLL gene rearrangement istranslocation of the MLL gene at 11q23. In some embodiments, the MLLgene rearrangement is a partial tandem duplication of the MLL gene.

The present invention also provides a method for treating cancer byadministering to a subject in need thereof a therapeutically effectiveamount of a compound of Formula (I) (i.e., Compound 2) or its N-oxide ora pharmaceutically acceptable salt thereof:

where the subject is unable to receive other therapy to treat the cancerdue to age or intercurrent illness.

In any methods described herein, the subject may be simultaneouslytreated with another therapy to treat acute myeloid leukemia, acutelymphoblastic leukemia, myelodysplastic syndrome, a myeloproliferativedisorder, or chronic myelogenous leukemia. For example, the anothertherapy is standard of care for the treatment of acute myeloid leukemia.For example, the another therapy is standard of care for the treatmentof acute lymphoblastic leukemia.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are respectively a table and a plot demonstrating thepotency and selectivity of the anti-proliferative activity of Compound 2using a panel of MLL-rearranged and non-MLL-rearranged human leukemiacell lines. The cell lines used in the study are listed in FIG. 1A.

FIG. 2 is a plot showing the tumor growth over 21 days of treatment withcontrol or Compound 2 at various dosages.

FIG. 3A is a plot showing the estimated steady state plasmaconcentrations of Compound 2 in Groups 4 and 5 as determined by theaveraged blood samples taken on days 7, 14 and 21.

FIG. 3B is a plot showing the Compound 2 plasma concentrations plottedagainst time after ip injection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and uses of a compound having thefollowing formula (I) (Compound 2) or its N-oxide or a pharmaceuticallyacceptable salt thereof for treating cancer, particularly for treatingleukemia:

The present invention provides methods for the treatment of leukemia ina subject in need thereof by administering to a subject in need of suchtreatment, a therapeutically effective amount of a compound of thepresent invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof. The leukemia can be acute orchronic leukemia. Preferably, the leukemia is acute myeloid leukemia,acute lymphocytic leukemia or mixed lineage leukemia. The presentinvention further provides the use of a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, for the preparation of a medicament usefulfor the treatment of leukemia. The leukemia can be acute or chronicleukemia. For example, the leukemia can be acute myeloid leukemia, acutelymphocytic leukemia or mixed lineage leukemia, myelodysplasticsyndrome, a myeloproliferative disorder, or chronic myelogenousleukemia. Exemplary myeloproliferative disorder includes, for example,but is not limited to, chronic myelogenous leukemia (CML), polycythemiavera (PV), essential thrombocytosis (ET), or myelofibrosis (MF).

The present invention provides methods for the treatment of a disease ordisorder mediated by rearrangements of the MLL gene in a subject in needthereof by administering to the subject in need of such treatment, atherapeutically effective amount of a compound of the present invention,or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof. In certain embodiments the MLL rearrangement is atranslocation of a gene on chromosome 11q23. In certain embodiments theMLL rearrangement is partial tandem duplication (MLL-PTD). The presentinvention further provides the use of a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, for the preparation of a medicament usefulfor the treatment of a disease or disorder mediated by MLL generearrangements.

As used herein, a “subject in need thereof” is a subject having cancer,or hematologic cancer or leukemia. For example, the leukemia is acutemyeloid leukemia, acute lymphoblastic leukemia, acute mixed lineageleukemia, myelodysplastic syndrome, a myeloproliferative disorder, orchronic myelogenous leukemia. For example, the subject has a leukemiainvolving translocation of the MLL gene at 11q23, MLL-PTD or an advancedhematologic malignancy. In certain embodiments the subject is thesubject is unable to receive other therapy to treat the cancer due toage or intercurring illness. In certain embodiments the subject is atleast 50 years old, or at least 60 years old, or at least 65 years old,or at least 70 years old or older.

In some embodiments, the subject in need thereof had at least one priortherapy to treat acute myeloid leukemia, acute lymphoblastic leukemia,acute mixed lineage leukemia, myelodysplastic syndrome, amyeloproliferative disorder, or chronic myelogenous leukemia.

In some embodiments, the subject has refractory cancer. Refractorycancer is a malignancy for which surgery is ineffective, which is eitherinitially unresponsive to chemo- or radiation therapy, or which becomesunresponsive over time.

In some embodiments, the subject has hematological cancer recurrencefollowing remission.

In some embodiments, the subject is not a candidate for allogeneic stemcell transplantation.

In some embodiments, the subject is simultaneously being treated withanother therapy to treat acute myeloid leukemia, acute lymphoblasticleukemia, acute mixed lineage leukemia, myelodysplastic syndrome, amyeloproliferative disorder, or chronic myelogenous leukemia. Additionaltherapies and agents that can be used to treat these cancers are knownto the skilled artisan; and are described in U.S. ProvisionalApplication No. 61/785,446, the contents of which are incorporatedherein by reference in their entireties. In certain embodiments thepatient is treated with the standard of care treatment as described inthe most current National Comprehensive Cancer Network (NCCN)guidelines. For example, for the treatment of AML such therapy mayinclude all-trans retinoic acid (ATRA), cytaribine (Ara-C),daunorubicin, idarubicine, arsenic trioxide (ATO, 6-mercaptopurineand/or methotrexate. For example, for the treatment of ALL the standardof care may include vincristine, corticosteroids such as prednisone,and/or methotrexate.

In some embodiments, the subject received and failed all known effectivetherapies for the hematological cancer that the subject has or issuffering from.

A “subject” includes a mammal. The mammal can be e.g., any mammal, e.g.,a human, primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat,camel, sheep or a pig. Preferably, the mammal is a human.

A cancer that is to be treated can be evaluated by DNA cytometry, flowcytometry, or image cytometry. A cancer that is to be treated can betyped as having 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cellsin the synthesis stage of cell division (e.g., in S phase of celldivision). A cancer that is to be treated can be typed as having a lowS-phase fraction or a high S-phase fraction.

As used herein, “monotherapy” refers to the administration of a singleactive or therapeutic compound to a subject in need thereof. Preferably,monotherapy will involve administration of a therapeutically effectiveamount of an single active compound. For example, cancer monotherapywith one of the compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, analog or derivative thereof, to asubject in need of treatment of cancer. In one aspect, the single activecompound is a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof.

As used herein, “treating” or “treat” describes the management and careof a patient for the purpose of combating a disease, condition, ordisorder and includes the administration of a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, to alleviate the symptoms or complicationsof a disease, condition or disorder, or to eliminate the disease,condition or disorder.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, can also beused to prevent a disease, condition or disorder. As used herein,“preventing” or “prevent” describes reducing or eliminating the onset ofthe symptoms or complications of the disease, condition or disorder.

As used herein, the term “alleviate” is meant to describe a process bywhich the severity of a sign or symptom of a disorder is decreased.Importantly, a sign or symptom can be alleviated without beingeliminated. In a preferred embodiment, the administration ofpharmaceutical compositions of the invention leads to the elimination ofa sign or symptom, however, elimination is not required. Effectivedosages are expected to decrease the severity of a sign or symptom. Forinstance, a sign or symptom of a disorder such as cancer, which canoccur in multiple locations, is alleviated if the severity of the canceris decreased within at least one of multiple locations.

As used herein, the term “severity” is meant to describe the potentialof cancer to transform from a precancerous, or benign, state into amalignant state. Alternatively, or in addition, severity is meant todescribe a cancer stage, for example, according to the TNM system(accepted by the International Union Against Cancer (UICC) and theAmerican Joint Committee on Cancer (AJCC)) or by other art-recognizedmethods. Cancer stage refers to the extent or severity of the cancer,based on factors such as the location of the primary tumor, tumor size,number of tumors, and lymph node involvement (spread of cancer intolymph nodes). Alternatively, or in addition, severity is meant todescribe the tumor grade by art-recognized methods (see, National CancerInstitute, www.cancer.gov). Tumor grade is a system used to classifycancer cells in terms of how abnormal they look under a microscope andhow quickly the tumor is likely to grow and spread. Many factors areconsidered when determining tumor grade, including the structure andgrowth pattern of the cells. The specific factors used to determinetumor grade vary with each type of cancer. Severity also describes ahistologic grade, also called differentiation, which refers to how muchthe tumor cells resemble normal cells of the same tissue type (see,National Cancer Institute, www.cancer.gov). Furthermore, severitydescribes a nuclear grade, which refers to the size and shape of thenucleus in tumor cells and the percentage of tumor cells that aredividing (see, National Cancer Institute, www.cancer.gov).

In another aspect of the invention, severity describes the degree towhich a tumor has secreted growth factors, degraded the extracellularmatrix, become vascularized, lost adhesion to juxtaposed tissues, ormetastasized. Moreover, severity describes the number of locations towhich a primary tumor has metastasized. Finally, severity includes thedifficulty of treating tumors of varying types and locations. Forexample, inoperable tumors, those cancers which have greater access tomultiple body systems (hematological and immunological tumors), andthose which are the most resistant to traditional treatments areconsidered most severe. In these situations, prolonging the lifeexpectancy of the subject and/or reducing pain, decreasing theproportion of cancerous cells or restricting cells to one system, andimproving cancer stage/tumor grade/histological grade/nuclear grade areconsidered alleviating a sign or symptom of the cancer.

As used herein the term “symptom” is defined as an indication ofdisease, illness, injury, or that something is abnormal in the body.Symptoms are felt or noticed by the individual experiencing the symptom,but may not easily be noticed by others. Others are defined asnon-health-care professionals.

As used herein the term “sign” is also defined as an indication thatsomething is abnormal in the body. But signs are defined as things thatcan be seen by a doctor, nurse, or other health care professional.

Treating cancer can result in a reduction in size of a tumor. Areduction in size of a tumor may also be referred to as “tumorregression”. Preferably, after treatment, tumor size is reduced by 5% orgreater relative to its size prior to treatment; more preferably, tumorsize is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75% or greater.Size of a tumor may be measured by any reproducible means ofmeasurement. The size of a tumor may be measured as a diameter of thetumor.

Treating cancer can result in a reduction in tumor volume. Preferably,after treatment, tumor volume is reduced by 5% or greater relative toits size prior to treatment; more preferably, tumor volume is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75% or greater. Tumor volume may bemeasured by any reproducible means of measurement.

Treating cancer results in a decrease in number of tumors. Preferably,after treatment, tumor number is reduced by 5% or greater relative tonumber prior to treatment; more preferably, tumor number is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75%. Number of tumors may bemeasured by any reproducible means of measurement. The number of tumorsmay be measured by counting tumors visible to the naked eye or at aspecified magnification. Preferably, the specified magnification is 2×,3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in a decrease in number of metastatic lesionsin other tissues or organs distant from the primary tumor site.Preferably, after treatment, the number of metastatic lesions is reducedby 5% or greater relative to number prior to treatment; more preferably,the number of metastatic lesions is reduced by 10% or greater; morepreferably, reduced by 20% or greater; more preferably, reduced by 30%or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75%. The number of metastatic lesions may be measured byany reproducible means of measurement. The number of metastatic lesionsmay be measured by counting metastatic lesions visible to the naked eyeor at a specified magnification. Preferably, the specified magnificationis 2×, 3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population receivingcarrier alone. Preferably, the average survival time is increased bymore than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population ofuntreated subjects. Preferably, the average survival time is increasedby more than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in increase in average survival time of apopulation of treated subjects in comparison to a population receivingmonotherapy with a drug that is not a compound of the present invention,or a pharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof. Preferably, the average survival time is increasedby more than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in a decrease in the mortality rate of apopulation of treated subjects in comparison to a population receivingcarrier alone. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to an untreatedpopulation. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to a populationreceiving monotherapy with a drug that is not a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,analog or derivative thereof. Preferably, the mortality rate isdecreased by more than 2%; more preferably, by more than 5%; morepreferably, by more than 10%; and most preferably, by more than 25%. Adecrease in the mortality rate of a population of treated subjects maybe measured by any reproducible means. A decrease in the mortality rateof a population may be measured, for example, by calculating for apopulation the average number of disease-related deaths per unit timefollowing initiation of treatment with an active compound. A decrease inthe mortality rate of a population may also be measured, for example, bycalculating for a population the average number of disease-relateddeaths per unit time following completion of a first round of treatmentwith an active compound.

Treating cancer can result in a decrease in tumor growth rate.Preferably, after treatment, tumor growth rate is reduced by at least 5%relative to number prior to treatment; more preferably, tumor growthrate is reduced by at least 10%; more preferably, reduced by at least20%; more preferably, reduced by at least 30%; more preferably, reducedby at least 40%; more preferably, reduced by at least 50%; even morepreferably, reduced by at least 50%; and most preferably, reduced by atleast 75%. Tumor growth rate may be measured by any reproducible meansof measurement. Tumor growth rate can be measured according to a changein tumor diameter per unit time.

Treating cancer can result in a decrease in tumor regrowth. Preferably,after treatment, tumor regrowth is less than 5%; more preferably, tumorregrowth is less than 10%; more preferably, less than 20%; morepreferably, less than 30%; more preferably, less than 40%; morepreferably, less than 50%; even more preferably, less than 50%; and mostpreferably, less than 75%. Tumor regrowth may be measured by anyreproducible means of measurement. Tumor regrowth is measured, forexample, by measuring an increase in the diameter of a tumor after aprior tumor shrinkage that followed treatment. A decrease in tumorregrowth is indicated by failure of tumors to reoccur after treatmenthas stopped.

Treating or preventing cancer can result in a decrease in the number orproportion of cells having an abnormal appearance or morphology.Preferably, after treatment, the number of cells having an abnormalmorphology is reduced by at least 5% relative to its size prior totreatment; more preferably, reduced by at least 10%; more preferably,reduced by at least 20%; more preferably, reduced by at least 30%; morepreferably, reduced by at least 40%; more preferably, reduced by atleast 50%; even more preferably, reduced by at least 50%; and mostpreferably, reduced by at least 75%. An abnormal cellular appearance ormorphology may be measured by any reproducible means of measurement. Anabnormal cellular morphology can be measured by microscopy, e.g., usingan inverted tissue culture microscope. An abnormal cellular morphologycan take the form of nuclear pleiomorphism.

Treating cancer or a cell proliferative disorder can result in celldeath, and preferably, cell death results in a decrease of at least 10%in number of cells in a population. More preferably, cell death means adecrease of at least 20%; more preferably, a decrease of at least 30%;more preferably, a decrease of at least 40%; more preferably, a decreaseof at least 50%; most preferably, a decrease of at least 75%. Number ofcells in a population may be measured by any reproducible means. Anumber of cells in a population can be measured by fluorescenceactivated cell sorting (FACS), immunofluorescence microscopy and lightmicroscopy. Methods of measuring cell death are as shown in Li et al.,Proc Natl Acad Sci USA. 100(5): 2674-8, 2003. In an aspect, cell deathoccurs by apoptosis.

As used herein, the term “selectively” means tending to occur at ahigher frequency in one population than in another population. Thecompared populations can be cell populations. Preferably, a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, acts selectively on a canceror precancerous cell but not on a normal cell. Preferably, a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, acts selectively to modulateone molecular target (e.g., a target protein methyltransferase) but doesnot significantly modulate another molecular target (e.g., a non-targetprotein methyltransferase). The invention also provides a method forselectively inhibiting the activity of an enzyme, such as a proteinmethyltransferase. Preferably, an event occurs selectively in populationA relative to population B if it occurs greater than two times morefrequently in population A as compared to population B. An event occursselectively if it occurs greater than five times more frequently inpopulation A. An event occurs selectively if it occurs greater than tentimes more frequently in population A; more preferably, greater thanfifty times; even more preferably, greater than 100 times; and mostpreferably, greater than 1000 times more frequently in population A ascompared to population B. For example, cell death would be said to occurselectively in cancer cells if it occurred greater than twice asfrequently in cancer cells as compared to normal cells.

Administering a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof, to acell or a subject in need thereof can result in modulation (i.e.,stimulation or inhibition) of an activity of a protein methyltransferaseof interest.

Administering a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof, to acell or a subject in need thereof results in modulation (i.e.,stimulation or inhibition) of an activity of an intracellular target(e.g., substrate). Several intracellular targets can be modulated withthe compounds of the present invention, including, but not limited to,protein methyltrasferase.

Activating refers to placing a composition of matter (e.g., protein ornucleic acid) in a state suitable for carrying out a desired biologicalfunction. A composition of matter capable of being activated also has anunactivated state. An activated composition of matter may have aninhibitory or stimulatory biological function, or both.

Elevation refers to an increase in a desired biological activity of acomposition of matter (e.g., a protein or a nucleic acid). Elevation mayoccur through an increase in concentration of a composition of matter.Treating cancer or a cell proliferative disorder can result in celldeath, and preferably, cell death results in a decrease of at least 10%in number of cells in a population. More preferably, cell death means adecrease of at least 20%; more preferably, a decrease of at least 30%;more preferably, a decrease of at least 40%; more preferably, a decreaseof at least 50%; most preferably, a decrease of at least 75%. Number ofcells in a population may be measured by any reproducible means. Anumber of cells in a population can be measured by fluorescenceactivated cell sorting (FACS), immunofluorescence microscopy and lightmicroscopy. Methods of measuring cell death are as shown in Li et al.,Proc Natl Acad Sci USA. 100(5): 2674-8, 2003. In an aspect, cell deathoccurs by apoptosis.

An effective amount of a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, is not significantly cytotoxic to normal cells. Atherapeutically effective amount of a compound is not significantlycytotoxic to normal cells if administration of the compound in atherapeutically effective amount does not induce cell death in greaterthan 10% of normal cells. A therapeutically effective amount of acompound does not significantly affect the viability of normal cells ifadministration of the compound in a therapeutically effective amountdoes not induce cell death in greater than 10% of normal cells. In anaspect, cell death occurs by apoptosis.

Contacting a cell with a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, can induce or activate cell death selectively in cancercells. Administering to a subject in need thereof a compound of thepresent invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, can induce or activate celldeath selectively in cancer cells. Contacting a cell with a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, can induce cell deathselectively in one or more cells affected by a cell proliferativedisorder. Preferably, administering to a subject in need thereof acompound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, induces celldeath selectively in one or more cells affected by a cell proliferativedisorder.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al.,Molecular Cloning, A Laboratory Manual (3^(rd) edition), Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (2000); Coligan et al., CurrentProtocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., CurrentProtocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., ThePharmacological Basis of Therapeutics (1975), Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 18^(th) edition (1990).These texts can, of course, also be referred to in making or using anaspect of the invention.

The present invention also provides pharmaceutical compositionscomprising a compound of the present invention, for example compoundhaving Formula (I) (Compound 2), in combination with at least onepharmaceutically acceptable excipient or carrier.

A “pharmaceutical composition” is a formulation containing the compoundsof the present invention in a form suitable for administration to asubject. In one embodiment, the pharmaceutical composition is in bulk orin unit dosage form. The unit dosage form is any of a variety of forms,including, for example, a capsule, an IV bag, a tablet, a single pump onan aerosol inhaler or a vial. The quantity of active ingredient (e.g., aformulation of the disclosed compound or salt, hydrate, solvate orisomer thereof) in a unit dose of composition is an effective amount andis varied according to the particular treatment involved. One skilled inthe art will appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, inhalational, buccal, sublingual, intrapleural,intrathecal, intranasal, and the like. Dosage forms for the topical ortransdermal administration of a compound of this invention includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In one embodiment, the active compound is mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers, or propellants that are required.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

A compound or pharmaceutical composition of the invention can beadministered to a subject in many of the well-known methods currentlyused for chemotherapeutic treatment. For example, for treatment ofcancers, a compound of the invention may be injected directly intotumors, injected into the blood stream or body cavities or taken orallyor applied through the skin with patches. The dose chosen should besufficient to constitute effective treatment but not as high as to causeunacceptable side effects. The state of the disease condition (e.g.,cancer, precancer, and the like) and the health of the patient shouldpreferably be closely monitored during and for a reasonable period aftertreatment.

The term “therapeutically effective amount”, as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic selected for administration.Therapeutically effective amounts for a given situation can bedetermined by routine experimentation that is within the skill andjudgment of the clinician. In a preferred aspect, the disease orcondition to be treated is cancer. In another aspect, the disease orcondition to be treated is a cell proliferative disorder.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

Dosage and administration are adjusted to provide sufficient levels ofthe active agent(s) or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug interaction(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

The pharmaceutical compositions containing active compounds of thepresent invention may be manufactured in a manner that is generallyknown, e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Pharmaceutical compositions may be formulated ina conventional manner using one or more pharmaceutically acceptablecarriers comprising excipients and/or auxiliaries that facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Of course, the appropriate formulation is dependentupon the route of administration chosen.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol and sorbitol, and sodium chloridein the composition. Prolonged absorption of the injectable compositionscan be brought about by including in the composition an agent whichdelays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblepharmaceutically acceptable carrier. They can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The active compounds can be prepared with pharmaceutically acceptablecarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

In therapeutic applications, the dosages of the pharmaceuticalcompositions used in accordance with the invention vary depending on theagent, the age, weight, and clinical condition of the recipient patient,and the experience and judgment of the clinician or practitioneradministering the therapy, among other factors affecting the selecteddosage. Generally, the dose should be sufficient to result in slowing,and preferably regressing, the growth of the tumors and also preferablycausing complete regression of the cancer. Dosages can range from about0.01 mg/kg per day to about 5000 mg/kg per day. In preferred aspects,dosages can range from about 1 mg/kg per day to about 1000 mg/kg perday. In an aspect, the dose will be in the range of about 0.1 mg/day toabout 50 g/day; about 0.1 mg/day to about 25 g/day; about 0.1 mg/day toabout 10 g/day; about 0.1 mg to about 3 g/day; or about 0.1 mg to about1 g/day, in single, divided, or continuous doses (which dose may beadjusted for the patient's weight in kg, body surface area in m², andage in years). An effective amount of a pharmaceutical agent is thatwhich provides an objectively identifiable improvement as noted by theclinician or other qualified observer. For example, regression of atumor in a patient may be measured with reference to the diameter of atumor. Decrease in the diameter of a tumor indicates regression.Regression is also indicated by failure of tumors to reoccur aftertreatment has stopped. As used herein, the term “dosage effectivemanner” refers to amount of an active compound to produce the desiredbiological effect in a subject or cell.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The compounds of the present invention are capable of further formingsalts. All of these forms are also contemplated within the scope of theclaimed invention.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the compounds of the present invention wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines, alkalior organic salts of acidic residues such as carboxylic acids, and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include, but are not limitedto, those derived from inorganic and organic acids selected from2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethanedisulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic,glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic,hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic,isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic,mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic,pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic,salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic,sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurringamine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.

Other examples of pharmaceutically acceptable salts include hexanoicacid, cyclopentane propionic acid, pyruvic acid, malonic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, muconic acid, and the like. The present invention also encompassessalts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates) or crystalforms (polymorphs) as defined herein, of the same salt.

The compounds of the present invention can also be prepared as prodrugs,for example, pharmaceutically acceptable prodrugs. The terms “pro-drug”and “prodrug” are used interchangeably herein and refer to any compoundwhich releases an active parent drug in vivo. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.), the compounds of thepresent invention can be delivered in prodrug form. Thus, the presentinvention is intended to cover prodrugs of the presently claimedcompounds, methods of delivering the same and compositions containingthe same. “Prodrugs” are intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when such prodrug is administered to a subject. Prodrugs in thepresent invention are prepared by modifying functional groups present inthe compound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds of the present invention wherein a hydroxy, amino,sulfhydryl, carboxy or carbonyl group is bonded to any group that may becleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl,free carboxy or free carbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, formates, phosphates, sulfates andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxy functional groups, esters (e.g., ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g., N-acetyl) N-Mannich bases, Schiff bases andenaminones of amino functional groups, oximes, acetals, ketals and enolesters of ketone and aldehyde functional groups in compounds of theinvention, and the like, See Bundegaard, H., Design of Prodrugs, p 1-92,Elesevier, New York-Oxford (1985).

The compounds, or pharmaceutically acceptable salts, esters or prodrugsthereof, are administered orally, nasally, transdermally, pulmonary,inhalationally, buccally, sublingually, intraperintoneally,subcutaneously, intramuscularly, intravenously, rectally,intrapleurally, intrathecally and parenterally. In one embodiment, thecompound is administered orally. One skilled in the art will recognizethe advantages of certain routes of administration.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counter,or arrest the progress of the condition.

Techniques for formulation and administration of the disclosed compoundsof the invention can be found in Remington: the Science and Practice ofPharmacy, 19^(th) edition, Mack Publishing Co., Easton, Pa. (1995). Inan embodiment, the compounds described herein, and the pharmaceuticallyacceptable salts thereof, are used in pharmaceutical preparations incombination with a pharmaceutically acceptable carrier or diluent.Suitable pharmaceutically acceptable carriers include inert solidfillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

All percentages and ratios used herein, unless otherwise indicated, areby weight. Other features and advantages of the present invention areapparent from the different examples. The provided examples illustratedifferent components and methodology useful in practicing the presentinvention. The examples do not limit the claimed invention. Based on thepresent disclosure the skilled artisan can identify and employ othercomponents and methodology useful for practicing the present invention.

In the synthetic schemes described herein, compounds may be drawn withone particular configuration for simplicity. Such particularconfigurations are not to be construed as limiting the invention to oneor another isomer, tautomer, regioisomer or stereoisomer, nor does itexclude mixtures of isomers, tautomers, regioisomers or stereoisomers.

Compounds described herein are assayed for modulation of activity, forexample, histone methylation, modulation of cell growth and/or IC₅₀,described in the examples below.

Compound DOT1L IC₅₀ (μM) 2 0.00074

Additional compounds suitable for the methods of the invention, as wellas pharmaceutical compositions and uses thereof, are described inWO2012/075381, WO2012/075492, WO2012/082436, and WO2012/75500, thecontents of each of which are hereby incorporated by reference in theirentireties.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES

Nuclear magnetic resonance (NMR) spectra were obtained on a BrukerAvance 400 operating at a field strength of 400.130 MHz or a Bruker DRX500 MHz NMR or HNMR spectra were obtained on a 500 MHz Bruker AVANCE IIIspectrometer. Common reaction solvents were either high performanceliquid chromatography (HPLC) grade or American Chemical Society (ACS)grade, and anhydrous as obtained from the manufacturer unless otherwisenoted. LCMS was performed on a Waters Micromass ZMD with a Waters 2795Separations Module and Waters 996 photodiode array detector and a WatersMicromass ZQ with a Waters 2695 Separations Module and Waters 996photodiode array detector or a Waters Micromass Platform LCZ singlequadrupole mass spectrometer with a Waters 600 solvent delivery module,Waters 515 ancillary pumps, Waters 2487 UV detector and a Gilson 215autosampler and fraction collector. Or, LCMS analysis was performedusing SQ mass spectrometer coupled to AGILENT 1200 Series HPLC. LCMSdata, where available, are provided in the examples below as well as inTable 1. The MS data are provided using the convention for m/z in theformat, [M+H]⁺.

The compounds of the present invention can be prepared using knownchemical transformations adapted to the particular situation at hand.Additional information can be found in WO2012/075381, WO2012/075492,WO2012/082436, and WO2012/75500, the contents of each of which arehereby incorporated by reference in their entireties.

Example 1 Synthesis of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol(Compound 2) Step 1: Synthesis of cis and trans methyl3-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)amino)cyclobutanecarboxylate

A solution of methyl 3-oxocyclobutanecarboxylate (4.60 g, 35.94 mmol),9-((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine(11.0 g, 35.94 mmol) and Ti(iPrO)₄ (4.0 g, 14.08 mmol) in MeOH (80 mL)was stirred at 45° C. for 2 h, then NaCNBH₃ (4.5 g, 71.87 mmol) wasadded. The reaction was stirred at RT overnight. The reaction wasquenched with aq. sat. NaHCO₃ (40 mL) and filtered, extracted with DCM(80 mL×3), dried over Na₂SO₄ and concentrated. The residue was purifiedby preparative-HPLC to obtain the title compound (6.2 g, Yield 41%). ¹HNMR (500 MHz, CDCl₃): δ_(H) 8.38-8.34 (m, 1H), 7.90 (s, 1H), 5.98 (d,J=3.0 Hz, 1H), 5.75 (br s, 2H), 5.48-5.46 (m, 1H), 5.03-5.01 (m, 1H),4.35-4.33 (m, 1H), 3.69-3.66 (m, 3H), 3.50-3.17 (m, 1H), 3.05-2.73 (m,3H), 2.48-2.44 (m, 2H), 1.95-1.91 (m, 2H), 1.62 (s, 3H), 1.39 (s, 3H)ppm; ESI-MS (m/z): 419.2[M+1]⁺.

The cis/trans mixture of methyl3-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)amino)cyclobutanecarboxylate(6.2 g) was separated via chiral HPLC (CHIRALCEL AD-H 20*250 mm, 5 um(Daicel), Column temperature: 35° C., Mobile phase: CO2/Methanol (0.1%DEA)=70/30, Flow rate: 50 g/min) to give the pure cis product (3.5 g)and pure trans product (1.7 g).

Step 2: Synthesis of (1S,3s)-methyl3-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)cyclobutanecarboxylate

To a solution of cis methyl3-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)amino)cyclobutanecarboxylate(2.0 g, 4.78 mmol) in CH₃CN (15 ml) was added 2-iodopropane (4.0 g,23.92 mmol) and K₂CO₃ (1.0 g, 7.18 mmol). The reaction was heated to 95°C. overnight in a sealed tube. The mixture was filtered, the filtratewas concentrated and purified by SGC (DCM: MeOH=12:1) to obtain thetitle compound (1.9 g, Yield 86%). ¹H NMR (500 MHz, CDCl₃): δ_(H) 8.37(s, 1H), 7.89 (s, 1H), 6.03 (d, J=1.5 Hz, 1H), 5.53-5.48 (m, 3H), 5.00(br s, 1H), 4.25 (brs, 1H), 3.66 (s, 3H), 3.19-3.18 (m, 1H), 2.96 (brs,1H), 2.80-2.78 (m, 1H), 2.67-2.58 (m, 2H), 2.20-2.12 (m, 4H), 1.62 (s,3H), 1.39 (s, 3H), 1.00 (d, J=6.0 Hz, 3H), 0.84 (d, J=6.0 Hz, 3H) ppm;ESI-MS (m/z): 461.4[M+1]⁺.

Step 3: Synthesis of(1S,3s)-3-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)cyclobutanecarbaldehyde

To a solution of (1S,3s)-methyl3-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)cyclobutanecarboxylate(1.2 g, 2.60 mmol) in DCM (50 ml) was added DIBAL-H dropwise at −78° C.until all the starting material was consumed as determined by TLC. MeOH(2 ml) was added and the mixture was stirred to RT for 30 min upon whichwater (50 ml) was added and the mixture was extracted with DCM (50ml×2). The organic layer was dried over Na₂SO₄ and concentrated toobtain crude title compound (1.0 g which was used) directly in the nextstep. ¹H NMR (500 MHz, CDCl₃): δ_(H) 9.56 (d, J=2.5 Hz, 1H), 8.36 (s,1H), 7.88 (s, 1H), 6.03 (d, J=2.5 Hz, 1H), 5.66 (br s, 2H), 5.50 (dd,J=2.0, 6.5 Hz, 1H), 5.01 (dd, J=3.5, 6.5 Hz, 1H), 3.331-3.337 (m, 1H),2.96-2.97 (m, 1H), 2.77-2.59 (m, 3H), 2.14-2.05 (m, 4H), 1.60 (s, 3H),1.39 (s, 3H), 1.01 (d, J=6.5 Hz, 3H), 0.85 (d, J=6.0 Hz, 3H) ppm.

Step 4: Synthesis of (E)-ethyl3-((1S,3s)-3-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)cyclobutyl)acrylate

To a solution of(1S,3s)-3-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)cyclobutanecarbaldehyde (930 mg, 2.16 mmol) in CH₃CN:DCM=5:1 (50 ml) was addedethyl 2-(diethoxyphosphoryl)acetate (484 mg, 2.16 mmol), DBU (328 mg,2.16 mmol) and LiCl (91 mg, 2.16 mmol). The mixture was stirred at RTfor 1 h and then concentrated. Water (20 ml) was added and the mixtureextracted with DCM (25 ml×3). The combined organic layers were driedover Na₂SO₄, concentrated and the residue was purified by SGC(DCM:MeOH=30:1) to obtain title compound (900 mg, Yield 83%). ¹H NMR(500 MHz, CDCl₃): δ_(H) 8.36 (s, 1H), 7.89 (s, 1H), 6.94-6.90 (m, 1H),6.03 (s, 1H), 5.72-5.89 (m, 1H), 5.57 (s, 2H), 5.52 (d, J=4.5 Hz, 1H),5.00 (dd, J=3.5, 6.0 Hz, 1H), 4.25 (d, J=3.0 Hz, 1H), 4.21-4.17 (m, 2H),3.14 (brs, 1H), 2.961-2.936 (m, 1H), 2.74-2.52 (m, 3H), 2.22-2.14 (m,2H), 1.79-1.76 (m, 2H), 1.60 (s, 3H), 1.40 (s, 3H), 1.30-1.27 (m, 3H),1.00 (d, J=7.0 Hz, 3H), 0.82 (d, J=6.5 Hz, 3H) ppm; ESI-MS (m/z):501.4[M+1]⁺.

Step 5: Synthesis of ethyl3-((1S,3r)-3-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)cyclobutyl)propanoate

To a solution of (E)-ethyl3-((1S,3s)-3-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)cyclobutyl)acrylate (900 mg, 1.8 mmol) in MeOH (50 ml) was added Pd/C (20 mg). Themixture was stirred at RT overnight under an atmosphere of hydrogen. Themixture was filtered and the filtrate was concentrated to obtain titlecompound (700 mg, Yield 78%). ¹H NMR (500 MHz, CDCl₃): δ_(H) 8.36 (s,1H), 7.89 (s, 1H), 6.03 (d, J=2.5 Hz, 1H), 5.69 (s, 2H), 5.51 (dd,J=2.5, 8.0 Hz, 1H), 4.99 (dd, J=4.0, 7.5 Hz, 1H), 4.26 (brs, 1H),4.13-4.08 (m, 2H), 2.99-2.92 (m, 2H), 2.706-2.655 (m, 1H), 2.539-2.486(m, 1H), 2.18-2.02 (m, 4H), 1.76 (brs, 1H), 1.65-1.60 (m, 5H), 1.43-1.37(m, 5H), 1.26-1.23 (m, 2H), 0.97 (d, J=9.0 Hz, 3H), 0.79 (d, J=8.5 Hz,3H) ppm; ESI-MS (m/z): 503.4[M+1]⁺.

Step 6: Synthesis of3-((1S,3r)-3-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)cyclobutyl)propanoicacid

To a solution of ethyl3-((1S,3r)-3-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)cyclobutyl)propanoate (650 mg, 1.29 mmol) inTHF:MeOH=5:1 (30 ml) was added LiOH.H₂O (543 mg, 1.29 mmol). The mixturewas stirred at RT overnight, concentrated and then taken up in MeOH (10ml). 1M HCl solution was added dropwise at 0° C. until pH=7. The mixturewas concentrated and purified with preparative-HPLC to give titlecompound (170 mg).

Step 7: Synthesis ofN-(2-amino-4-(tert-butyl)phenyl)-3-((1S,3r)-3-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)cyclobutyl)propanamide

To a solution of3-((1S,3r)-3-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)cyclobutyl)propanoicacid (170 mg, 0.36 mmol) in DCM (15 ml) was added4-tert-butylbenzene-1,2-diamine (117 mg, 0.72 mmol), EDCl (137 mg, 0.72mmol), HOBT (97 mg, 0.72 mmol) and TEA (217 mg, 2.15 mmol). The mixturewas stirred at RT overnight and concentrated. Saturated NaHCO₃ solution(20 ml) was added and the mixture extracted with DCM (20 ml×3). Theorganic layers were dried over Na₂SO₄ and concentrated. The crude waspurified with preparative-TLC (DCM:MeOH=12:1) to afford the titlecompound (110 mg crude).

Step 8: Synthesis of9-((3aR,4R,6R,6aR)-6-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine

A solution ofN-(2-amino-4-(tert-butyl)phenyl)-3-((1S,3r)-3-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)cyclobutyl)propanamide(110 mg) in AcOH (10 ml) was heated to 65° C. overnight. The mixture wasconcentrated, saturated NaHCO₃ solution (20 ml) was added and themixture extracted with DCM (20 ml×3). The combined organic layers weredried over Na₂SO₄ and concentrated to give the title compound (105 mgcrude). ¹H NMR (500 MHz, CDCl₃): δ_(H) 8.36 (s, 1H), 7.89 (s, 1H),7.48-7.24 (m, 3H), 6.01 (d, J=1.5 Hz, 1H), 5.60-5.53 (m, 3H), 4.98 (dd,J=3.0, 6.5 Hz, 1H), 4.22 (brs, 1H), 2.97 (brs, 1H), 2.874-2.847 (m, 1H),2.56-2.50 (m, 3H), 1.87-1.78 (m, 2H), 1.70-1.54 (m, 7H), 1.35-1.17 (m,14H), 0.90 (d, J=6.5 Hz, 3H), 0.80 (d, J=6.5 Hz, 3H) ppm; ESI-MS (m/z):603.5[M+1]⁺.

Step 9: Synthesis of Compound 2

A solution of9-((3aR,4R,6R,6aR)-6-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine(105 mg) in HCl/MeOH (2.5 mol/L) (10 mL) was stirred at RT for 2 h, thenconcentrated to dryness. K₂CO₃ (96 mg) in water (0.5 mL) and MeOH (5 mL)were added and the resulting mixture was stirred for another 10 min atRT and then filtered. The filtrate was concentrated and the residue waspurified by preparative-HPLC (xbridge 30 mm*150 mm, Mobile phase:A:water (10 mM NH4HCO3) B: CAN, Gradient: 35-45% B in 10 min, 45-45% Bin 6 min, stop at 20 min, Flow rate: 50 ml/min) to give Compound 2 (50mg, yield: 51%) as a white solid. ¹HNMR (500 MHz, MeOD): δ_(H) 8.29 (s,1H), 8.20 (s, 1H), 7.47-7.39 (m, 3H), 5.96 (d, J=4.0 Hz, 1H), 4.70-4.75(m, 1H), 4.26-4.27 (m, 1H), 4.05-4.06 (m, 1H), 3.140-3.155 (m, 1H),3.00-2.76 (m, 5H), 2.18-2.16 (m, 2H), 1.87-1.85 (m, 2H), 1.57-1.55 (m,2H), 1.36 (s, 9H), 1.01 (d, J=6.5 Hz, 3H), 0.94 (d, J=6.5 Hz, 3H) ppm;ESI-MS (m/z): 563.4 [M+1]⁺.

Example 2 Bioassay Protocol and General Methods

Cell Culture.

Human hematological tumor cell lines THP-1, RS4; 11, and MV4-11 wereobtained from ATCC, MOLM-13 cells were obtained from DSMZ. All lineswere grown in RPMI 1640 containing 10% FBS and maintained using thevendors recommended cell densities and environmental conditions. Mediawas supplemented with non essential amino acids and L-Glutamine. THP-1cells were also supplemented with 0.05 mM (3-Mercaptoethanol.

Methylation Analysis.

Cells were seeded at 5×10⁵ cells/mL in a 12 well plate at a final volumeof 2 mLs Cells were dosed with compounds to the appropriateconcentration from a 50 mM DMSO stock solution. Compound and media wererefreshed every two days over the course of seven day incubation bycounting cells using trypan blue exclusion (Vicell), pelleting at 200 gfor 5 minutes and resuspending in fresh media containing compound at afinal cell concentration of 5×10⁵ cells/mL. Following compoundincubation, histones were extracted from 1×10⁶ cells using a commercialhistone extraction kit (Active Motif). Purified histones werequantitated using the BCA protein assay (Pierce) with a BSA standardcurve. 400 ng of isolated histones were fractionated by SDS-PAGE on a4-20% gel and transferred to nitrocellulose membranes. Membranes wereincubated with various primary and secondary antibodies and imaged onthe Licor imaging system (Odyssey). The H3K79-Me2 rabbit polyclonal waspurchased from Abcam. Other rabbit polyclonal antibodies includingH3K4-Me3, H3K9-Me3, H3K27-Me2, and H3K27-Me3 were purchased from CellSignaling Technologies (CST). A mouse monoclonal total H3 antibody wasused as a loading control (CST). Fluorescently labeled secondaryantibodies were purchased from Odyssey.

Cell Growth and Viability Analysis.

Cells were harvested from exponentially growing cell cultures and seededat 3×10⁴ cells per well. Samples were maintained in a 96 well blackwalled clear bottom plate (Corning). A final concentration of 50 uMcompound in 0.2% DMSO was added to the appropriate wells on Day 0.Treatment of MV4-11 and MOLM-13 lasted 14 days, while THP-1 cells weretreated for 18 days. Compound and media were replaced every two daysduring incubation by transferring samples to a V-bottom plate (Corning),spinning at 200 g for 5 minutes in a room temperature rotor,resuspending in fresh media containing compound and transferring back tothe assay plate. Cells were counted periodically using the GuavaViacount assay and read on the EasyCyte Plus instrument (Millipore).Assay plates were split when necessary to within recommended celldensities. Final cell counts were adjusted to take cell splits intoaccount and reported as total viable cells/well.

HOXA9 (qPCR).

Cells were treated with compound for 7 days similar to methylationassay. Cell were pelleted at 200 g in a room temperature rotor and totalRNA isolated using the Qiagen RNeasy kit. RNA concentration and qualitywas determined by using the Nanovue (GE Healthcare). Total RNA wasreverse transcribed using a high capacity cDNA reverse transcription kit(Applied Biosystems). A predesigned labeled primer set for HOXA9 waspurchased from Applied Biosystems. qPCR reactions contained 50 ng cDNA,1× labeled primer and 1× Taqman universal PCR master mix (AppliedBiosystems). Samples were run on a 7900 HT Fast Real Time PCR machine(Applied Biosystems) with PCR conditions of 2 min 50° C., 10 min 95° C.,40 cycles at 15 sec 95° C. and 1 min 60° C. HOXA9 cycle numbers werenormalized to the house keeping gene B2 microglobulin (B2M predesignedcontrol from Applied Biosystems). Percent of DMSO control was calculatedwith the equation, percent control=(2̂^(−ΔΔCT))*100 where the ΔΔCT is thedifference between normalized HOXA9 sample and control (ΔCT sample−ACTcontrol=ΔΔCT).

Determination of IC₅₀.

Test compounds were serially diluted 3 fold in DMSO for 10 points and 1μl was plated in a 384 well microtiter plate. Positive control (100%inhibition standard) was 2.5 uM final concentration ofS-adenosyl-L-homocysteine and negative control (0% inhibition standard)contained 1 μl of DMSO. Compound was then incubated for 30 minutes with40 μl per well of DOT1L (1-416) (0.25 nM final concentration in assaybuffer: 20 mM TRIS, pH 8.0, 10 mM NaCl, 0.002% Tween20, 0.005% BovineSkin Gelatin, 100 mM KCl, and 0.5 mM DTT). 10 μl per well of substratemix (same assay buffer with 200 nM S-[methyl-³H]-adenosyl-L methionine,600 nM of unlabeled S-[methyl-³H]-adenosyl-L methionine, and 20 nMoligonucleosome) was added to initiate the reaction. Reaction wasincubated for 120 minutes at room temperature and quenched with 10 μlper well of 100 μM S-methyl-adenosyl-L methionine. For detection,substrate from 50 μl of reaction was immobilized on a 384 wellStreptavidin coated Flashplate (Perkin Elmer) (also coated with 0.2%polyethyleneimine) and read on a Top Count scintillation counter (PerkinElmer).

Example 3 Tumor Anti-Proliferation Assays

In Vitro Anti-Proliferative Assay.

The potency and selectivity of the anti-proliferative activity of thecompounds of the present invention were assessed using a panel ofMLL-rearranged and non-MLL-rearranged human leukemia cell lines. Thecell lines used in the study are listed in FIG. 1A. The MLL-rearrangedpanel included cell lines derived from ALL, AML and biphenotypicleukemias harboring MLL-AF4, MLL-AF9 or MLL-ENL fusions. These celllines recruit DOT1L. The panel also included five cell lines that do notpossess an MLL-rearrangement, and one cell line that bears a partialtandem duplication of the MLL gene (MLL-PTD).

Exponentially growing cells were plated, in triplicate, in 96-wellplates at a density of 3×10⁴ cells/well in a final volume of 150 μl.Cells were incubated in the presence of increasing concentrations ofCompound 2. Anti-proliferative activity was determined by cell viabilitymeasurements every 3-4 days for up to 14 days. On days of cell counts,growth media and Compound 2 were replaced and cells split back to adensity of 5×10⁴ cells/well.

The half maximal inhibitory concentration (IC₅₀) results in FIG. 1 showthat Compound 2 demonstrates potent nanomolar anti-proliferativeactivity against three of four MLL-rearranged cell lines tested (MV4; 11(MLL-AF4), MOLM-13 (MLL-AF9), and KOPN-8 (MLL-ENL). EOL-1 cells whichexpress an MLL-PTD were also highly sensitive to Compound 2 (IC₅₀=11nM). RS4; 11 cells and two non MLL-rearranged cells (Reh and Kasumi-1)were 1-3 log orders less sensitive, and two non-MLL-rearranged cells(Jurkat and HL-60) showed no activity. Overall, the results indicatethat Compound 2 potently and selectively inhibits the proliferation ofMLL-rearranged leukemia cell lines and a subset of non-MLL-rearrangedleukemia cell lines.

In Vivo Anti-Proliferative Assay.

The in vivo anti-tumor activity of the compounds of the presentinvention were assessed in a mouse xenograft model of MLL-rearrangedleukemia.

Four groups of 20 (Groups 1, 3, 4 and 5), and one group of 8 (Group 2)female nude mice (average weight of 0.023 kg) bearing MV4-11 xenografttumors of sizes ranging from 80-120 mm³ were implanted subcutaneouslywith minipumps (Alzet Model 2001). Group 1 received vehicle only fromthe pump. Group 2 received vehicle only from the pump plus ip injectionstid (8 hours apart) of vehicle. Group 3 received 112 mg/kg/day from thepump plus ip injections tid (8 hours apart) of 20 mg/kg of Compound 2for a total daily dose of 172 mg/kg/day. Group 4 and 5 received 112 and56 mg/kg/day of Compound 2 from the pumps, respectively. Pumps weredesigned to last for 7 days and were exchanged twice to give totalinfusion duration of 21 days exposure.

A single blood sample was taken from all animals in Groups 4 and 5 ondays 7, 14, and 21 and assayed for plasma levels of Compound 2. Bloodsamples were taken from Group 3 on days 7 and 14 at the following timepoints (3 animals per time point): 5 minutes pre-ip dose, and 15 min, 30min, 1, 2, and 4 hours post ip dose. On day 21 three hours after thelast ip injection, a single blood sample was taken from Group 3. Tumorsize was measured every 4 days. After 21 days the study was terminated,and mean TGI calculated.

FIG. 2 shows the tumor growth over the 21 days of dosing. There was nodifference in tumor size between the two vehicle control groups. Thehigh dose minipump group supplemented with ip dosing showed astatistically significant TGI of >70% compared to the controls. The 56and 112 mg/kg/day groups showed non-statistically significant TGI valuesof 43 and 38%, respectively, compared to controls. Compound 2 isreferred to as Ex. 2 in FIG. 2.

FIG. 3A shows the estimated steady state plasma concentrations ofCompound 2 in Groups 4 and 5 as determined by the averaged blood samplestaken on days 7, 14, and 21. The data suggest that the average steadystate Compound 2 plasma levels ranged from 99 to 152 ng/ml for Group 4,and 52 to 238 ng/ml for Group 5. The average plasma level from the lastsampling on day 21 was 99 ng/ml for Group 4 and 52 ng/ml for Group 5.

FIG. 3B shows the Compound 2 plasma concentrations plotted against timeafter ip injection. The ip injections produced a significant increase inplasma exposure to Compound 2 in terms of both the C_(max) (4200 to 5000ng/ml) after each of the tid injections and the daily AUCs over thoseproduced by the steady state plasma level resulting from the continuousinfusion. Overall, the results indicate that Compound 2 demonstratedsignificant anti-tumor activity in a mouse xenograft model ofMLL-rearranged leukemia.

Example 4 A First-in-Human Phase 1 and Expanded Cohort Study of Compound2 in Advanced Hematologic Malignancies, Including Acute Leukemia withRearrangement of the MLL Gene

A study is carried out to determine the safe dose of Compound 2, toevaluate the safety of Compound 2 in patients with advanced hematologicmalignancies, and to conduct a preliminary assessment of theanti-leukemia activity of Compound 2 in patients with acute leukemiasbearing rearrangements of the MLL gene. Conditions for this studyinclude Acute Myeloid Leukemia, Acute Lymphoblastic Leukemia,Myelodysplastic Syndrome, A myeloproliferative disorder, and ChronicMyelogenous Leukemia.

Details of the study are shown in the table 1 below.

TABLE 1 Study Design. Designated Measure Time Frame as safety issueAnalysis Primary The maximum tolerated dose up to 12 yes The MTD isdefined as one Outcome (MTD) of Compound 2 as months dose level belowthe level Measure determined by incidence of in which >1 dose-limitingprotocol-specified dose- adverse events (as defined limiting adverseevents by the protocol) are observed. Secondary Pharmacokinetic profileup to 12 no Analysis of Cmax, AUC and Outcome of Compound 2 monthssteady state concentration Measures The type, incidence, and up to 24yes Evaluation of adverse events, severity of adverse events monthsvital signs, physical examina- in patients treated with tion, 12-leadECG, and labo- Compound 2 ratory assessments Anti-leukemic activity ofup to 24 no Evaluation of response by Compound 2 in patients with monthsstandard criteria for AML or acute leukemia harboring ALL aMLL-rearrangement Effects of Compound 2 on up to 24 no / histone H3K79methylation months in peripheral blood mononuclear (PBMC) and leukemiacells; and target gene expression in leukemia cells.

A subset of patients with acute myeloid leukemia (AML) and acutelymphoblastic leukemia (ALL) harbor rearrangements of the MLL gene,which are detected either by cytogenetic or fluorescent in-situhybridization evaluation at the time of diagnosis. DOT1L plays animportant role in the malignant process in these leukemias, and Compound2 is a molecule that blocks the activity of DOT1L.

Compound 2 is administered as a 21-day continuous intravenous infusion,and safe dosages are assessed in patients with hematologic malignancies.The study will have two phases. The first phase will assess escalatingdoses of Compound 2 in order to determine the maximally tolerated dose(MTD) of Compound 2. Once the MTD is established, a second phase of thestudy will further evaluate the safety of Compound 2 and assess theanti-leukemia activity of Compound 2 in MLL-rearranged leukemia.

Arms Assigned Interventions Experimental: Drug: Compound 2 Compound 2Dose- Dose-escalation, 21-day continuous IV infusion of escalation andeach 28-day cycle. Number of cycles: until disease extension cohortsprogression or unacceptable toxicity develops.

Eligibility for Patients

-   1. Ages Eligible for Study: 18 Years and older-   2. Genders Eligible for Study: Both

Inclusion Criteria:

-   1. Male and female patients aged≧18 years.-   2. AML, ALL, acute mixed lineage leukemia, myelodysplastic syndrome    (International Prognostic Scoring System Int-2 or high-risk),    myeloproliferative disorder, or chronic myelogenous leukemia meeting    the following criteria (only patients with acute leukemia with    rearrangement of the MLL gene will be eligible for the expanded    cohort):    -   At least one prior therapy;    -   Refractory disease on most recent therapy, or disease recurrence        following remission on most recent therapy;    -   Received and failed all known effective therapies for their        disease; and/or    -   Not a candidate for allogeneic stem cell transplantation.-   3. Eastern Cooperative Oncology Group (ECOG) performance status of    0-2.-   4. Patients must have the following clinical laboratory values:    -   Serum creatinine≦2 mg/dL or creatinine clearance>60 mL/minute;    -   Total bilirubin≦1.5 times the ULN for the institution, unless        considered due to Gilbert's syndrome;    -   ALT or AST≦twice the upper limit of normal (ULN), unless        considered due to organ leukemic involvement;    -   Absolute neutrophil count≧1,000/μL (unless due to documented        leukemic involvement of the bone marrow at the time of study        entry);    -   Platelets≧100,000/μL (unless due to documented leukemic        involvement of the bone marrow at the time of study entry); and    -   PT or aPTT<1.5 times the ULN.-   5. Able and willing to give written informed consent.-   6. Life expectancy of at least 3 months.

Exclusion Criteria:

-   1. Uncontrolled intercurrent illness or psychiatric illness/social    situations that would limit compliance with study requirements.-   2. Active heart disease.-   3. Receiving any other standard treatment for their hematologic    malignancy.-   4. Receiving strong CYP3A4 inhibitors/inducers.-   5. Known history of cerebrovascular accident in the past 6 months.-   6. Known bleeding diathesis.-   7. Known, active involvement of the central nervous system by    leukemia.-   8. On immunosuppressive therapy.-   9. Known active infection.-   10. Pregnant or nursing females.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

EQUIVALENTS

The invention can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A method for treating cancer comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound of Formula (I) or its N-oxide or a pharmaceuticallyacceptable salt thereof:

wherein the subject had at least one prior therapy to treat ahematological cancer.
 2. The method of claim 1, wherein thehematological cancer is refractory to the prior therapy.
 3. The methodof claim 1, wherein the hematological cancer shows recurrence followingremission.
 4. The method of claim 1, wherein the subject received andfailed all known effective therapies for the hematological cancer. 5.The method of claim 1, wherein the hematological cancer is selected fromthe group consisting of acute myeloid leukemia, acute lymphoblasticleukemia, myelodysplastic syndrome, a myeloproliferative disorder, andchronic myelogenous leukemia.
 6. The method of claim 1, wherein thehematological cancer is leukemia.
 7. The method of claim 6, wherein thesubject has a leukemia characterized by MLL gene rearrangement.
 8. Themethod of claim 7, wherein the MLL gene rearrangement is translocationof the MLL gene at 11q23.
 9. The method of claim 7, wherein the MLL generearrangement is a partial tandem duplication of the MLL gene.
 10. Amethod for treating cancer comprising administering to a subject in needthereof a therapeutically effective amount of a compound of Formula (I)or its N-oxide or a pharmaceutically acceptable salt thereof:

wherein the subject is unable to receive other therapy to treat thecancer due to age or intercurrent illness.
 11. The method of claim 1,wherein the subject is simultaneously being treated with another therapyto treat acute myeloid leukemia, acute lymphoblastic leukemia,myelodysplastic syndrome, a myeloproliferative disorder, or chronicmyelogenous leukemia.
 12. The method of claim 11, wherein the anothertherapy is standard of care for the treatment of acute myeloid leukemia.13. The method of claim 11, wherein the another therapy is standard ofcare for the treatment of acute lymphoblastic leukemia.